Phosphorylation code of human nucleophosmin includes four cryptic sites for hierarchical binding of 14-3-3 proteins

• DOI: 10.1101/2024.02.13.580064
• Publication Date: 2024-02-14T04:30:30Z
• Authors: Anna A. Kapitonova; Kristina V. Perfi...; Richard B. Cooley; Nikolai N. Sluchanko
• Keywords: Nucleophosmin; NPM1; Phosphopeptide

Abstract Nucleophosmin (NPM1) is the 46th most abundant human protein with many functions whose dysregulation leads to various cancers. Pentameric NPM1 resides in nucleolus but can also shuttle cytosol. regulated by multisite phosphorylation, yet molecular consequences of site-specific phosphorylation remain elusive. Here we identify four 14-3-3 binding sites concealed within its oligomerization and α-helical C-terminal domains that are found phosphorylated vivo . By combining mutagenesis, in-cell PermaPhos technology for site-directed incorporation a non-hydrolyzable phosphoserine mimic, show how promotes monomerization partial unfolding, recruit dimers low-micromolar affinity. Using fluorescence anisotropy quantified pairwise interactions all seven isoforms recombinant phosphopeptides assessed their druggability fusicoccin. This revealed complex hierarchy affinities toward primary (S48, S293) secondary (S106, S260) sites, differentially modulated small molecule. As three these phospho-sites reside signal sequences, this work highlights key mechanism regulation which control nucleocytoplasmic shuttling. It provides further evidence phosphorylation-induced structural rearrangements globular proteins serve expose otherwise cryptic 14-3-3-binding important cellular function.